The long term objective of this laboratory is to understand at the cellular level the alterations of insulin and glucagon action in the liver of patients with NIDDM. This effort is possible because a) we have a population of patients with and without NIDDM from whom intraoperative liver biopsies can be obtained, and b) we have methods to study in vitro hormone-responsive liver preparations. We have shown that the efficiency of lactate/pyruvate conversion to glucose is enhanced in NIDDM. Therefore, we will test the hypothesis that the balance between insulin and glucagon action regulating PEPCK expression is responsible for this alteration and contributes to fasting hyperglycemia. Since changes in glucokinase and GLUT-2 protein have been demonstrated in the liver in NIDDM, we will also study the regulation of these two important proteins (glucose sensors?) and test the hypothesis that their alterations contribute to postprandial hyperglycemia. These two hypotheses provide the focus of Specific Aim 1. Specific Aim 2 is based on the previous demonstration that the liver insulin-stimulated tyrosine kinase and the cross talk between the insulin receptor and Gi protein(s) are abnormal in NIDDM. We will test the hypothesis that protein kinase C (PKC) is responsible for the phosphorylation and inactivation of both the insulin receptor and Gi protein(s). PKC activity should be increased in NIDDM because a) glucose stimulates de novo synthesis of diacylglycerol (DAG), and b) in the liver phosphatidylinositol 4,5-bis-phosphate (PIP2) specific phospholipase C (PLC) activity is increased, which also increases DAG and activates PKC. Our preliminary data indicate that glucagon is responsible for the increase in PIP2 PLC activity. These results imply that in NIDDM glucagon might not only be involved in the mechanism of increased glucose production, but also in the induction of insulin resistance. This is the hypothesis to be tested in Specific Aim 3. Toward this end, a putative positive pool containing the cDNA of the glucagon receptor has been isolated for use in detailed studies of glucagon action in NIDDM. It is hoped that this work will contribute to our understanding of the altered carbohydrate metabolism in NIDDM and help to develop a more rational therapeutic approach to the disease.